This project continues our long term goal of investigating the molecular mechanisms of membrane-acting antibiotics and understanding how they work in bacteria. These antibiotics utilize mechanisms different from conventional antibiotics and have already contributed to the campaign against the growing public health problem of antibiotic-resistant infections. Our specific aim 1 is to study the molecular mechanism of daptomycin. Daptomycin is the first FDA approved member of a new structural class of antibiotics that target the cytoplasmic membranes of Gram-positive pathogens. Unlike the host-defense antimicrobial peptides, daptomycin does not form transmembrane pores. Thus, despite its clinical use, the mechanism of daptomycin is still unknown. Using a method of aspirating membrane vesicles, we found daptomycin capable of extracting lipid molecules from membranes. We hypothesize that this is a so-far unrecognized bactericidal mechanism. We propose to investigate the lipid extracting effect of daptomycin by further developing the membrane aspirating methods. We will also use X-ray methods made available by new synchrotron radiation facilities to investigate the structures of daptomycin bound to a membrane. Our specific aim 2 proposes to close the gap between two different methods of studying membrane-acting antibiotics, i.e., the biophysical methods that use lipid bilayers in place of cell membranes and the direct studies with bacteria. Direct studies with bacteria are complicated by the outer cell wall and the periplasmic peptidoglycan. Our proposed method is to use bacterial spheroplasts that have been stripped off the cell wall and peptidoglycan. We will apply physical methods to the cytoplasmic membranes of spheroplasts to measure the elastic properties of bacterial membranes for the first time. We will study the direct interactions of membrane-acting antibiotics on spheroplast membranes and compare with the results on lipid bilayers. These results will connect the molecular mechanisms deduced from the lipid-bilayer studies to the actions on bacterial membranes. PHS 398/2590 (Rev. 06/09) Page Continuation Format Page